Research will be conducted to understand how G proteins are activated in the cytoplasm of the cell by a protein factor called Ric-8A. Heterotrimeric G proteins modulate cell metabolism, secretion, electrical conductivity, gene transcription, cell division and cellular motility, and therefore are essential to life in the doman of eukaryotes to which humans belong. Misregulation of G proteins is associated with cancer and a range of other diseases of relevance to general medicine. While most processes controlled by heterotrimeric G proteins occur at cell membranes, recent research has shown that G alpha subunits (G?) also control certain events in cell cytoplasm. Important among these is asymmetric cell division, which is essential for embryonic development. Ric-8A is critical regulator of G? in this process. Ric-8A is a Guanine nucleotide Exchange Factor (GEF) that catalyzes the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) at the active site of G?. This reaction transitions G? to the so-called activated state. In preliminary studies, the Principal Investigator's laboratory demonstrated that Ric-8A induces and stabilizes G?i1 (a Gi-class G? subunit) in a structurally heterogeneous or molten globule-like state. The nucleotide-free Ric-8A:G?i1 intermediate is stable in the absence of GTP. The hypothesis to be tested is that Ric-8A catalyzes nucleotide exchange by altering the global and local structure of G?i1, and by increasing flexibility and inducing dynamic behavior. The overall goal of the project is to characterize the trajectory of the exchange reaction from binding of Ric-8A to G?i1.GDP, to the formation of the nucleotide-free Ric-8A:G?i1 complex. The first two aims of the proposal address, respectively, the structure and the dynamic behavior of the Ric- 8A:G?i1 complex. The first aim is to use Double Electron-Electron Resonance (DEER) spectroscopy to observe large-scale changes in the structures of G?i1 and Ric-8A upon formation of the G?i1:Ric-8A complex. Hydrogen-Deuterium eXchange, followed by proteolysis and Mass Spectrometry (HDXMS) will be employed to observe structural changes at a level of detail that approaches single amino acid residues. In the second aim, Forster Resonance Energy Transfer (smFRET) studies of individual molecules, either freely diffusing or immobilized on a solid matrix, will be conducted to determine whether formation of the G?i1:Ric-8A complex is accompanied by changes in the dynamic behavior of either protein. The third aim of the proposal is to understand the mechanism by which Ric-8A alters the structure of G?i1 in the transition from the GDP- bound state to the nucleotide-free state. A combination of DEER and HDXMS methods will be used to study this transition. Site Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy, together with mutational scanning experiments, will be used to identify Ric-8A residues that are critical to G?i1-binding and GEF activity, and the sites at which Ric-8A makes contact with G?i1.